Hydroxycitric acid is an alpha-hydroxy tribasic acid (1,2-dihydroxypropane-1,2,3-tricarboxylic acid) with two asymmetric centers, hence the formation of two pairs of diasteroisomers or four different isomers: (−)hydroxycitric acid (I), (+)hydroxycitric acid (II), (−)allo-hydroxycitric acid (III), and (+)allo-hydroxycitric acid (IV). (1–2) The (−)hydroxycitric acid (HCA) isomer is found in the rind of Garcinia cambogia fruit (fam. Clusiaceae). (1–2) This isomer has been shown to be a potent linear competitive inhibitor of ATP citrate lyase enzyme, in vitro, demonstrating a much greater affinity for the purified enzyme than its natural substrate citrate as well as the other stereoisomers of hydroxycitric acid. (1–2) The biological importance of ATP citrate lyase is as a citrate cleavage enzyme which catalyzes the extramitochondrial cleavage of citrate to acetyl CoA and oxaloacetate, and facilitates the biosynthesis of fatty acids. The reversible inhibition of citrate lyase by (−) HCA may lead to the reduction of fatty acids synthesis and lipogenesis. These effects have been measured and demonstrated in vivo following the oral, intravenous or intraperitoneal administration of (−)hydroxycitrate to experimental animals. (3) When (−) HCA was given orally before the feeding period, the animals fed (−) HCA consumed less food and their hepatic synthesis of fatty acids and cholesterol was significantly diminished as compared to the untreated controls. (3–4) The observed decrease in food intake may be only one of the factors responsible for the (−) HCA promoted weight loss, because experimentation with rats fed (−) HCA showed weight loss with no decrease in cumulative food intake. (5) It seems that the potential mechanism of weight loss with (−) HCA may include an energy expenditure component, the nature of which remains undetermined. (5) This mechanism of energy expenditure, decreased lipogenesis, and the reduction in food intake in (−) HCA-treated animals may result in loss of weight and total body fat content. (6)
Although the potential of (−)HCA as a weight lowering compound has been recognized since the 1970's, only few clinical studies have been conducted with this compound. (7–12). These few studies examining HCA-mediated prevention of excess body fat, resulted in contradictory results, most likely due to HCA being poorly bioavailable in the cytosol of a target cell. In one clinical study of HCA, a controversial high fiber diet was used. The use of a high-fiber diet in combination with HCA may reduce gastrointestinal absorption of HCA, since high-fiber diets are known to reduce absorption of many nutrients and micronutrients. This issue becomes critical with HCA because its reported efficacy in inhibiting the intracellular enzyme, adenosine triphosphate (ATP)-citrate-lyase, depends entirely on the presence of HCA inside the target cell.
In their U.S. Patent, the Inventors addressed an important issue regarding the bioavailability of the HCA compounds. The U.S. Pat. No. 5,783,603 patent described a manufacturing process leading to a unique structure for a potassium salt of HCA, which facilitated its transport across biological membranes, effectively delivering more HCA into the cytosol for the competitive inhibition of ATP citrate lyase. Although the '603 patent related to an HCA compound having considerably improved bioavailability, its bioavailability was still relatively inefficient. For example, an in vitro study done on hepatic cells, indicates that 5 mM of extracellular potassium HCA could inhibit ATP citrate lyase. However, only 0.5 mM of potassium HCA is actually needed in the cytosol to effectively inhibit ATP citrate lyase. Therefore, a 10-fold excess amount of potassium HCA is needed outside of the target cell in order to achieve a concentration of 1/10 that amount in the cytosol. This finding of relatively poor bioavailability of HCA, was confirmed in pre-clinical experiments (14), and points out the need to further improve the bioavailability and efficacy of HCA.
Garcinol, like HCA, is isolated from Garcinia sp. fruit rind, and it exhibits anti-oxidant and chemoprotective properties (15). In one experiment, rats fed a garcinol diet (0.01% and 0.05%) showed a significantly reduced development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) as compared to control animals. Feeding of garcinol significantly elevated liver glutathione S-transferase, quinone reductase activities, suppressed O2- and NO generation and expression of iNOS and COX-2 proteins. These findings suggest a possible chemopreventive mechanism of garcinol.
Garcinol and isogarcinol were evaluated for their antibacterial activity against methicillin-resistant Staphylococcus aureus (16). These compounds showed a minimum inhibitory concentration at 3.1–12.5 micrograms/ml, or nearly equal to that of the antibiotic, vancomycine.
In 1981, Krishnamurthy et al. (17) reported the isolation of garcinol, and its colorless isomer, isogarcinol, from Garcinia indica. Their structures were proposed on the basis of chemical and spectral data. Garcinol, C38H50O6, m.p. 122(o), crystallized out from the hexane extract of the fruit rind of G. indica as yellow needles (1.5 percent). The UV spectrum of garcinol suggested that the 1,3-diketone system is conjugated to the 3,4-dihydroxybenzoyl moiety. The IR spectrum of the trimethyl ether showed the presence of a saturated carbonyl group (1727 cm−1) and two oe,beta-unsaturated carbonyl groups (1668 and 1642 cm−1).
Isogarcinol was isolated by column chromatography of the extract. Its identity was established by mixed m.p. and by comparison of LN, IR, and PMR spectra. The IR spectrum of isogarcinol indicated the presence of saturated carbonyl group (1715 cm−1), an aroyl group (1670 cm−1) and an oe,beta-unsaturated carbonyl group (1635 cm−1).
Rao et al. (18) reported the isolation of cambogin (33H55O6), m.p. 242–244, from the latex of Garcinia cambogia tree. The structure was confirmed by UV, IR and NMR studies. UV: 231–234, 275–278 and 305–313; IR (Kbr): 1720 (saturated carbonyl), 1680 and 1642 cm−1 (unsaturated carbonyl and double bond). Besides cambogin they also reported the isolation of camboginol and related its structure to cambogin.
In 1982, N. Krishnamurthy et al. (19) isolated anthocyanin pigments from the fresh red ripe fruits of Kokam (Garcinia indica). The rind portion was separated from the rest of the fruit and was macerated in a blender using methanol containing one percent HCl for three times. The extracts were combined, filtered and concentrated in vacuo at 30° C. Paper chromatography of the Kokam pigment extract showed two anthocyanin bands. The slower moving band was designated as B1 and the other B2. The total anthocyanin concentration was estimated to be 2.4 percent on a dry weight basis; the ratio of B1 to B2 is 1:4.
Anthocyanin B1 was identified as cyanidine-three-glucoside by chemical and spectroscopic studies. This compound on hydrolysis gave cyaniding and glucose. The UV spectral maximum (527 nm) of the glycosides shifted to 567 nm with aluminium chloride indicating that 3′- and 4′-hydroxyl groups of the cyaniding are free. The structure was confirmed by direct comparison with a sample of cyaniding-three-glucoside obtained from mulberry.
Anthocyanin B2 was identified as cyaniding-three-sambubioside. This anthocyanin on complete hydrolysis gave cyaniding, glucose and xylose. The spectral data suggested that B2 is a three-substituted glycoside of cyaniding. Hydrogen peroxide hydrolysis removed the disaccharide from the pigment which on further acid hydrolysis gave glucose and xylose. The structure was confirmed by direct comparison with a sample of cyaniding-3-sambubioside isolated from Roselle.
The present invention is based on the unexpected finding that combining HCA with natural compounds obtained from Garcinia sp. plant including garcinol (polyisoprenylated benzophenone) and/or anthocyanin compounds, results in not only an enhancement of the biological activity of HCA but also that of garcinol and/or anthocyanin.